Nitrous oxide (N2O) is widely used in clinical dentistry because of its anxiolytic and analgesic properties and because it is a weak anesthetic. It is also used in medicine as an adjunct to other anesthetics. Because N2O has euphorogenic properties, there is a potential for abuse. The recreational use of N2O has increased in recent years and health professionals are especially vulnerable because N2O is easily available and the dangers of dependence are not well recognized. Because N2O produces effects similar to opioids, benzodiazepines and ethanol, mechanisms responsible for opioid, ethanol, and benzodiazepine dependence may be involved in N2O dependence. In the present proposal, we will test the hypothesis that N2O withdrawal is the result of N20-induced release of endogenous opioid peptides and subsequent dependence to the endogenous opioid peptides. Our long range goals are also to test the role of non-opioid mechanisms (i.e. mechanisms important in benzodiazepine and ethanol dependence). N2O dependence is generally measured in mice by the appearance of handling induced convulsions (HIC) after exposure to and removal from N2O. We will develop methods for evaluating dependence by identifying and measuring HIC and other N2O withdrawal signs, particularly those typically associated with opioid withdrawal. Because there are differences between mouse strains in sensitivity and responses to N2O and opioids, several mouse strains will be used. We will also test the hypothesis that the kappa and delta opioid receptor subtypes are involved in the development of dependence in response to N2O since these opioid receptor subtypes are involved in N2O-induced analgesia. We will test these hypotheses by 1) examining opioid antagonist precipitated N2O withdrawal using the nonselective opioid antagonist, naloxone, and by using antagonists selective for mu, delta, and kappa opioid subtypes, 2) examining opioid subtype selective agonist protection against N2O withdrawal, and 3) examining N2O withdrawal after alkylation of mu, delta, and kappa opioid receptors. We expect these results to increase the understanding of mechanisms which underlie dependence to N2O and, because of the similarities between N2O dependence and opioid, ethanol, and benzodiazepine dependence, to understand the mechanisms underlying dependence to a variety of drugs of abuse.